Myeloperoxidase (MPO) is a critical component of the most important oxygen-dependent microbicidal system in human polymorphonuclear neutrophils (PMNs). Hereditary deficiency of MPO is a common disorder of human PMNs, occurring in 1 in 2000 of the population. Although the molecular basis for this disorder is not established, our prior work supports the hypothesis that a posttranslational defect results in the phenotype of MPO deficiency. According to this hypothesis, the genetic defect in MPO deficiency results in biosynthesis of a form of proMPO which is not processed normally into the peroxidatively active subunits of native MPO. In agreement with this hypothesis, we have shown that individuals whose PMNs are deficient in MPO activity contain a form of proMPO which is immunochemically closely related to normal proMPO. The goals of the proposed studies are to define the determinants of normal targeting of proMPO to lysosomes and to test the hypothesis that the inherited form of MPO deficiency is due to a defect in posttranslational processing of an aberrant proMPO. We propose two complementary approaches to achieve these goals. First, we will characterize normal processing of MPO using two analytical approaches. We will use antibodies to synthetic peptides derived from sequences in the proregion of MPO in order to isolate, characterize, and localize intracellularly proMPO and its cleavage products in cultured human promyelocytic leukemia cells. In addition, we will use Xenopus oocytes as a model system for studying MPO processing. We have cloned a full length cDNA for MPO into an SP6 transcription system. Capped transcripts will be generated, injected into frog oocytes, and characteristics of processing and intracellular transport will be studied. Subsequent studies will be performed using clones containing modified cDNAs as the inserts. Second, we will isolate, subclone, and sequence the extra Bgl II fragment which we have recently identified in most patients with the phenotype of MPO deficiency. In this way it will be possible to define the genotypic variability underlying MPO deficiency and to identify the site(s) in the gene where the critical mutation occurs. The proposed work will provide insight into sorting, processing, and intracellular targeting of the myeloid lysosomal protein MPO, the deficiency of which is likely the most common inherited disorder of human PMNs.